Screening for conditions of enhanced production of a recombinant beta-glucanase secreted into the medium by Escherichia coli.

The extracellular production of a hybrid bacterial beta-glucanase using Escherichia coli was studied by using combinations of promoters of varying strength for both a beta-glucanase as the target protein and the Kil protein as the releasing factor. Four strains with different combinations of promoter strengths were cultivated in shake-flasks on four different media to assess the cross-influence of promoter and medium in a general manner. Promoters were taken from natural as well as synthetic sequences known to exhibit either weak or strong promoter strength. By far the highest extracellular glucanase activity (>200 U ml(-1)) was achieved when a strain harbouring the kil gene under control of a strong synthetic stationary-phase promoter and the glucanase gene under control of a strong synthetic constitutive promoter was cultivated on a complex medium mainly composed of casein peptone, yeast extract, and glycerol.

Integrated bioprocess for the production and purification of recombinant proteins by affinity chromatography in Escherichia coli.

In order to improve the effectiveness of the production of recombinant proteins in E. coli, integrated fermentation processes were developed. Therefore, expression vectors were constructed containing a strongly expressed gene for a beta-glucanase fused with a metal-chelating affinity tag and a leader peptide for directing the fusion protein into the periplasmic space. Its export into the medium was achieved by means of co-expression of a bacteriocin-release protein, the Kil protein from pColE1. Bioreactors were modified so that special devices containing metal chelate pentadentate chelator PDC resins were located within the bioreactor. Using the bioreactor with an internal device the Zn2+-PDC had a 4.3-fold higher binding capacity than metal-free PDC (12.3 and 2.6 kU ml(-1) PDC, respectively. Using the bioreactor with charged PDC in an external circuit revealed even higher beta-glucanase concentration (65.6 kU ml(-1)), i.e. 1.5-fold compared to the internal adsorbent system.

Factors that influence the extracellular expression of streptavidin in Escherichia coli using a bacteriocin release protein.

Aiming to increase production of recombinant streptavidin in Escherichia coli, the effect of different leader sequences, different promoter strengths of the bacteriocin release protein (kil), host strain and medium composition on the expression and secretion into the medium was investigated. Expression vectors containing an expression or secretion unit were constructed with different combinations of leader sequence for the streptavidin gene and promoters for the kil gene and streptavidin gene. Results showed that a high-level extracellular production of streptavidin could be accomplished with E. coli BL21(DE3) by using the leader sequence of the phoA gene, a strong stationary-phase promoter for the kil gene and supplementation of the medium by glycine. Using a stationary-phase promoter for the expression of streptavidin had a negative effect.

Increasing the secretion ability of the kil gene for recombinant proteins in Escherichia coli by using a strong stationary-phase promoter.

By using a beta-glucanase from Bacillus as a model protein, we investigated whether the secretion competence based on the action of the kil gene can be improved using stronger promoters for the expression of the kil gene. Since the production of extracellular target proteins also depends on the promoter strengths of the target gene, we constructed four expression vectors with all possible combinations of a weak and a strong stationary-phase promoter for the kil gene, and a weak and a strong constitutive promoter, respectively, for the beta-glucanase gene. The results of batch fermentations showed that the use of stronger promoters generally decreased the cell density. However, a drastic increase of productivity of the cells to produce and secrete beta-glucanase resulted in a significantly higher activity of extracellular beta-glucanase. The yield of extracellular beta-glucanase can be increased (to 168 %) by using a strong promoter for the beta-glucanase alone. However, the increase was much higher when the weak promoter of the kil gene was replaced by a strong stationary-phase promoter (to 221 %). An even higher yield of extracellular beta-glucanase was reached when beta-glucanase was expressed by a strong promoter in addition indicating a combinatorial effect. This shows that the extracellular production of a recombinant target gene can be optimized by tuning the promoter strengths of components, the kil gene and the target gene.

The sequence upstream of the -10 consensus sequence modulates the strength and induction time of stationary-phase promoters in Escherichia coli.

We constructed a library of synthetic stationary-phase promoters for Escherichia coli. For designing the promoters, the known -10 consensus sequence, as well as the extended -10 region, and an A/T-rich region downstream of the -10 region were kept constant, whereas sequences from -37 to -14 were partially or completely randomised. For detection and selection of stationary-phase promoters, green fluorescent protein (GFP) with enhanced fluorescence was used. To establish the library, 33 promoters were selected, which differ in strength from 670 to more than 13,000 specific fluorescence units, indicating that the strength of promoters can be modulated by the sequence upstream of the -10 region. DNA sequencing revealed a preferential insertion of nucleotides depending on the position. By expressing the promoters in an rpoS-deficient strain, a special group of stationary-phase promoters was identified, which were expressed exclusively or preferentially by RNA polymerase holoenzyme Esigma(s). The DNA sequence of these promoters differed significantly in the region from -25 to -16. Furthermore, it was shown that the DNA curvature of the promoter region had no effect on promoter strength. The broad range of promoter activities make these promoters very suitable for fine-tuning of gene expression and for cost-effective large-scale applications in industrial bioprocesses.

Libraries of synthetic stationary-phase and stress promoters as a tool for fine-tuning of expression of recombinant proteins in Escherichia coli.

Due to their induction characteristics stationary-phase promoters have a great potential in biotechnological processes for the production of heterologous proteins on a large-scale. In order to broaden the utility of stationary-phase promoters in bacterial expression systems and to create novel promoters induced by metabolic conditions, a library of synthetic stationary-phase/stress promoters for Escherichia coli was constructed. For designing the promoters the known -10 consensus sequence as well as the extended -10 region and an A/T-rich region downstream of the -10 region were kept constant, while sequences from -37 to -14 were partially or completely randomized. For detection and selection of stationary-phase promoters GFP with enhanced fluorescence was used. The expression pattern of the GFP reporter system was compared with that of the LacZ reporter system. To screen and characterize colonies containing stationary-phase/stress promoters a bioinformatic approach was developed. In total, 33 promoters were selected which cover a broad range of promoter activities and induction times indicating that the strength of promoters can be modulated by partially randomizing the sequence upstream of the -10 region. The induction ratio of synthetic promoters at the transition from exponential to stationary-phase was from 4 to over 6000 and the induction time relative to the entrance into stationary-phase from -1.4 to 2.7 h. Ninety-one percentage of the promoters had no or only low background activity during exponential growth. The broad variability of the promoters offers good possibilities for fine-tuning of gene expression and for applications in industrial bioprocesses.